Fluorescent conjugates for analysis of molecules and cells

ABSTRACT

Sensitive detection techniques and compositions for such techniques are provided by employing fluorescent proteins having bilin prosthetic groups as labels. The bilin containing proteins can be conjugated to ligands or receptors for use in systems involving ligand-receptor binding for the analysis, detection or separation of ligands and receptors. Particularly, one or more of the bilin containing proteins may be used as labels in conjunction with each other or other fluorescers for defining subsets of naturally occurring aggregations e.g. cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation application of U.S. application Ser. No. 701,324,filed Feb. 13, 1985, which is a Continuation-in-part application of U.S.application Ser. No. 454,768, filed Dec. 30, 1982, now U.S. Pat. No.4,520,110, issued May 28, 1985, which was a Continuation-in-partapplication of U.S. application Ser. No. 309,169, filed Oct. 6, 1981,now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Fluorescent probes are valuable reagents for the analysis and separationof molecules and cells. Some specific examples of their application are:(1) identification and separation of subpopulations of cells in amixture of cells by the techniques of fluorescence flow cytometry,fluorescence-activated cell sorting, and fluorescence microscopy; (2)determination of the concentration of a substance that binds to a secondspecies (e.g., antigen-antibody reactions) in the technique offluorescence immunoassay; (3) localization of substances in gels andother insoluble supports by the techniques of fluorescence staining.These techniques are described by Herzenberg et al., "CellularImmunology," 3rd ed., chapt. 22, Blackwell Scientific Publications, 1978(fluorescence-activated cell sorting); and by Goldman, "FluorescenceAntibody Methods," Academic Press, New York, 1968 (fluorescencemicroscopy and fluorescence staining).

When employing fluorescers for the above purposes, there are manyconstraints on the choice of the fluorescer. One constraint is theabsorption and emission characteristics of the fluorescer, since manyligands, receptors, and materials associated with such compounds in thesample in which the compounds are found e.g. blood, urine, cerebrospinalfluid, will fluoresce and interfere with an accurate determination ofthe fluorescence of the fluorescent label. Another consideration is theability to conjugate the fluorescer to ligands and receptors and theeffect of such conjugation on the fluorescer. In many situations,conjugation to another molecule may result in a substantial change inthe fluorescent characteristics of the fluorescer and in some cases,substantially destroy or reduce the quantum efficiency of thefluorescer. A third consideration is the quantum efficiency of thefluorescer. Also of concern is whether the fluorescent molecules willinteract with each other when in close proximity, resulting inself-quenching. An additional concern is whether there is non-specificbinding of the fluorescer to other compounds or container walls, eitherby themselves or in conjunction with the compound to which thefluorescer is conjugated.

The applicability and value of the methods indicated above are closelytied to the availability of suitable fluorescent compounds. Inparticular, there is a need for fluorescent substances that emit in thelonger wavelength visible region (yellow to red). Fluorescein, a widelyused fluorescent compound, is a useful emitter in the green. However,the conventional red fluorescent label rhodamine has proved to be lesseffective than fluorescein. The impact of this deficiency is felt in thearea of fluorescence-activated cell sorting. The full potential of thispowerful and versatile tool has not yet been realized because oflimitations in currently available fluorescent tags. Two andthree-parameter fluorescence sorting have not been effectivelyexploited, largely because of the unavailability of good long wavelengthemitting probes.

Other techniques, involving histology, cytology, immunoassays would alsoenjoy substantial benefits from the use of a fluorescer with a highquantum efficiency, absorption and emission characteristics at longerwavelengths, having simple means for conjugation and being substantiallyfree of non-specific interference.

SUMMARY OF THE INVENTION

Proteins with bilin prosthetic groups are employed as fluorescent labelsin systems involving ligand-receptor reactions. The biliproteins arereadily conjugated, provide for high quantum efficiency with absorptionand emission at long wavelengths in the visible, and enhance thesensitivity and accuracy of methods involving ligand-receptor reactions.The biliproteins may be used individually, in combination, or togetherwith non-proteinaceous fluorescers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the high pressure liquid chromatograms of FIG. 1(a)phycoerythrin-immunoglobulin conjugate (PE-S-S-IgG) and the reactantprecursors thereof, FIG. 1(b) thiolated phycoerythrin (PE-SH) and FIG.1(c) activated immunoglobulin (IgG-S-S-Pyr).

FIG. 2 shows the fluorescence-activated cell sorter analysis of a cellpopulation containing spleen cells bearing PE-B-A stained anti-IgGimmunoglobulin.

FIG. 3a shows the visualization of a mixture of agarose beads containinga labeled anti-immunoglobulin by fluorescence microscopy utilizingstandard fluorescein emission filter combinations, wherein some beadswere labeled with PE-B-A and some beads were labeled withfluorescein-avidin.

FIG. 3b shows the visualization of the same cell population as in FIG.3a under fluorescence microscopy utilizing a red filter combination.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Compositions are provided comprising biliproteins, (the term"biliproteins" is equivalent to the term "phycobiliproteins") conjugatedto a member of a specific binding pair, said pair consisting of ligandsand receptors. These compositions find use for labeling by non-covalentbinding to the complementary member of the specific binding pair. A widevariety of methods involve competitive or non-competitive binding ofligand to receptor for detection, analysis or measurement of thepresence of ligand or receptor. Many of these techniques depend upon thepresence or absence of fluorescence as a result of non-covalent bindingof the labeled member of the specific binding pair with itscomplementary member.

The conjugates of the subject invention are biliproteins bound eithercovalently or non-covalently, normally covalently, to a particularligand or receptor. The biliproteins have a molecular weight of at leastabout 30,000 d, (d-daltons) more usually at least about 40,000 d, andmay be as high as 600,000 or more daltons usually not exceeding about300,000 d.

The biliproteins will normally be comprised of from 2 to 3 differentsubunits, where the subunits may ranged from about 10,000 to about60,000 molecular weight. The biliproteins are normally employed asobtained in their natural form from a wide variety of algae andcyanobacteria. The presence of the protein in the biliproteins providesa wide range of functional groups for conjugation to proteinaceous andnon-proteinaceous molecules. Functional groups which are present includeamino, thio and carboxy. In some instances, it may be desirable tointroduce functional groups, particularly thio groups where thebiliprotein is to be conjugated to another protein.

Depending upon the nature of the ligand or receptor to be conjugated, aswell as the nature of the biliprotein, the ratio of the two moietieswill vary widely, where there may be a plurality of biliproteins to oneligand or receptor or a plurality of ligands or receptors to onebiliprotein. For small molecules, that is, of molecular weight less than2,000 d, there will generally be on the average at least one and notmore than about 100, usually not more than about 60 conjugated to abiliprotein. With larger molecules, that is at least about 2,000molecular weight, more usually at least about 5,000 molecular weight,the ratio of biliproteins to ligand or receptor may vary widely, since aplurality of biliproteins may be present in the conjugate or a pluralityof the specific binding pair member may be present in the conjugate. Inaddition, in some instances, complexes may be formed by covalentlyconjugating a small ligand to a biliprotein and then forming a specificbinding pair complex with the complementary receptor, where the receptormay then serve as a ligand or receptor in a subsequent complex.

The ligand may be any compound of interest for which there is acomplementary receptor. For the most part, the ligands of interest willbe compounds having physiological activity, either naturally occurringor synthetic. One group of compounds will have molecular weights in therange of about 125 to 2,000, more usually from about 125 to 1,000, andwill include a wide variety of drugs, small polypeptides, vitamins,enzyme substrates, coenzymes, pesticides, hormones, lipids, etc. Thesecompounds for the most part will have at least one heteroatom, normallychalcogen (oxygen or sulfur) or nitrogen and may be aliphalitic,alicyclic, aromatic, or heterocyclic or combinations thereof.Illustrative compounds include epinephrine, prostaglandins, thyroxine,estrogen, corticosterone, ecdysone, digitoxin, aspirin, pencillin,hydrochlorothiazide, quinidine, oxytocin, somatostatin,diphenylhydantoin, retinol, vitamin K, cobalamin, biotin and folate.

Compounds of greater molecular weight, generally being 5,000 or moremolecular weight include poly(amino acids)-polypeptides andproteins-polysaccharides, nucleic acids, and combinations thereof e.g.glycosaminoglycans, glycoproteins, ribosomes, etc. Illustrativecompounds include albumins, globulins, hemogloblin, surface proteins oncells, such as T- and B-cells e.g. Leu, Thy, Ia, tumor specificantigens, α-fetoprotein, retinol binding protein, C-reactive protein,enzymes, toxins, such as cholera toxin, diphtheria toxin, botulinustoxin, snake venom toxins, tetrodotoxin, saxitoxin, lectins, such asconcanavalin, wheat germ agglutinin, and soy bean agglutinin,immunoglobulins, complement factors, lymphokines, mucoproteins,polysialic acids, chitin, collagen, keratin, etc.

Depending upon the molecule being labeled, a wide variety of linkinggroups may be employed for conjugating the biliprotein to the othermolecule. For the most part, with small molecules, those under 2,000molecular weight, the functional group of interest for linking will becarbonyl, either an aldehyde to provide for reductive amination or acarboxyl, which in conjunction with carbodiimide or as an activatedester e.g. N-hydroxy succinimide, will form a covalent bond with theamino groups present in the biliprotein; a thio ether or disulfide,where the biliprotein may be modified with an activated olefin and amercapto group added or activated mercapto groups joined e.g. Ellman'sreagent; isothiocyanate; diazonium; nitrene or carbene. Where thebiliproteins are conjugated with a protein, various bifuctional reagentsmay be employed, such as dialdehydes, tetrazolium salts, diacids, or thelike, or alternatively, one or both of the two proteins involved may bemodified for conjugation to the other protein, for example, a mercaptogroup may be present or be introduced on one protein and an activatedolefin e.g. maleimide introduced onto the other protein.

There is ample literature for conjugating a wide variety of compounds toproteins. See for example A. N. Glazer, The Proteins, Vol. IIA, 3rd Ed.,N. Neurath and R. L. Hill, eds., Academic Press, pp. 1-103 (19760; A. N.Glazer et al., "Chemical Modification of Proteins," LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 4, PRT I, T. S.Work and E. Work, eds., North-Holland Publishing Co. (1975); and K.Peters et al., Ann. Rev. Biochem., 46, 423-51 (1977), the descriptionsof which are incorporated by reference herein. Examples of commerciallyavailable cross-linking reagents are disclosed in the Pierce 1981-82Handbook and General Catalog, pp. 161-166, Pierce Chemical Co.,Rockford, Ill.

Known linking procedures as described in the above publications may beemployed. For example, the phycobiliprotein may be reacted withiminothiolane, thereby placing an accessible sulfhydryl group thereon.The other component of the conjugate may be activated by reaction withsuccinimidylpyridylthiopropionate. Mixture of the two preparedcomponents of the conjugate results in joining thereof through disulfidebonds. Alternatively, instead of employingsuccinimidylpyridylthiopropionate, the protein may be reacted withm-maleimidobenzoyl N-hydroxysuccinimide ester, and the resultingconjugate combined with the sulfhydry modified protein to form athioether.

As previously indicated, instead of having a covalent bond between thespecific binding pair member of interest and the biliprotein,non-covalent bonds may be employed. For example, if one wishes toconjugate a biliprotein to avidin, biotin may be covalently conjugatedto the biliprotein through its carboxyl group, and the resultingbiotinylated biliprotein combined with avidin, whereby a biliproteinlabeled avidin will result.

As already indicated, biliproteins are naturally occurring compoundswhich may be found in a wide variety of sources and even individualsources may have more than one biliprotein.

Examples of phycobiliproteins useful in the present invention areallophycocyanin, phycocyanin, phycoerythrin, allophycocyanin B,B-phycoerythrin, phycoerythrocyanin, and b-phycoerythrin. The structuresof phycobiliproteins have been studied and their fluorescent spectralproperties are known. See A. N. Glazer, "Photosynthetic AccessoryProteins with Bilin Prosthetic Groups," Biochemistry of Plants, Volume8, M. D. Hatch and N. K. Boardman, EDS., Academic Press, pp. 51-96(1981), and A. N. Glazer, "Structure and Evolution of PhotosyntheticAccessory Pigment Systems with Special Reference to Phycobiliproteins,"The Evolution of Protein Structure and Function, B. S. Sigman and M. A.Brazier, EDS., Academic Press, pp. 221-244 (1980). The spectroscopicproperties, including fluorescence emission maxima, of some commonphycobiliproteins are shown below in Table 1.

                  TABLE 1                                                         ______________________________________                                        SPECTROSCOPIC PROPERTIES OF                                                   PHYCOBILIPROTEINS                                                                                 Absorption    Fluorescence                                                    maxima in     emission                                                Distri- the visible.sup.2                                                                           maximum.sup.2                               Biliprotein bution.sup.1                                                                          (nm)          (nm)                                        ______________________________________                                        Allophycocyanin B                                                                         C,R     671 > 618     680                                         Allophycocyanin                                                                           C,R     650           660                                         C-Phycocyanin                                                                             C,R     620           637                                         R-Phycocyanin                                                                             R       617 > 555± 636                                         Phycoerythrocyanin                                                                        C       568 > 590(s)  607                                         C-Phycoerythrin                                                                           C       565           577                                         b-Phycoerythrin                                                                           R       545 > 563(s)  570                                         B-Phycoerythrin                                                                           R       545 > 563 > 498(s)                                                                          575                                         R-Phycoerythrin                                                                           C,R     567 > 538 > 498                                                                             578                                         ______________________________________                                         .sup.1 C = cyanobacteria; R = red algae.                                      .sup.2 For a given biliprotein, the exact positions of the absorption and     emission maxima vary somewhat depending on the organism that serves as th     source of the protein and on the method of purification.                 

Of particular interest are biliproteins having absorption maxima of atleast about 450 nm, preferably at least about 500 nm, having Stokesshifts of at least 15 nm, preferably at least about 25nm, and havingfluorescence emission maxima of at least about 500 nm, preferably atleast about 550 nm. The subject conjugates may be used in a wide varietyof ways, enhancing known methodologies for the detection, diagnosis,measurement and study of antigens, either present as individualmolecules, or in more complex organizations, such as viruses, cells,tissue, organelles e.g. plastids, nuclei, etc.

One of the uses of the subject conjugates is in fluorescent staining ofcells. The cells may then be observed under a microscope, the presenceof the fluorescer being diagnostic of the presence of a specificdeterminant site or the cells may be used in a fluorescence activatedcell sorter (FACS). One or more of the biliproteins may be used, wherethe fluorescence emission maximum of the biliproteins is separated by atleast about 15 nm, preferably by at least about 25 nm. Alternatively,the biliproteins may be used in conjunction with fluorescers other thanbiliproteins, for examples fluorescein, dansyl, umbelliferone,benzoxadiazoles, pyrenes, rose bengal, etc., where the emission maximaare separated by at least about 15 nm, preferably at least about 25 nm.

By using combinations of fluorescers, one can provide for the detectionof subsets of aggregations, such as particular types of cells, strainsof organisms, strains of viruses, the natural complexing or interactionof different proteins or antigens, etc. Combinations of particularinterest are combinations of fluorescein with biliproteins capable ofbeing activated with the same laser light source. That is, biliproteinswhich have absorption maxima in the range of about 450 to 500 nm e.g.phycoerythrin.

Another use of the subject biliproteins is in immunoassays orcompetitive protein, binding assays, where the subject biliproteinsserve as fluorescent labels. Here, the biliprotein may be conjugated toeither a ligand or a receptor, particularly an antibody. While for themost part the antibodies will be IgG, other antibodies such as IgA, IgD,IgE and IgM may also find use. In addition, various naturally occurringreceptors may be employed, particularly receptors having high bindingspecificity, such as avidin. By biotinylating either the receptor, thebiliprotein or both, one can link various molecules through avidin. Awide variety of fluorescent assays are known. A few of these assays areillustrated in U.S. Pat. Nos. 3,998,943; 3,985,867; 3,996,345;4,036,946; 4,067,959; 4,160,016 and 4,166,105, the relevant portions ofwhich are incorporated herein by reference.

The biliproteins have many favorable properties. (1) they have very highabsorption coefficients in the longer wavelength visible spectralregion; (2) they have high fluorescence quantum yields; (3) they arestable proteins and have good storage stability; (4) they are highlysoluble in aqueous solutions; (5) the biliprotein unit can readily becoupled to a wide range of biologically specific molecules; (6) they donot bind non-specifically to cells. The fluorescence ofbiliprotein-biomolecule conjugates is more than thirty times as intenseas that of fluorescein conjugates, on a molar basis. The long wavelengthemitting fluorescent conjugates of the present invention have anadditional advantage over shorter wavelength emitters. Most biomoleculesin cells and body fluids do not absorb and emit in the red end of thevisible spectrum. Consequently, biliprotein conjugates are less subjectto interference by endogenous biomolecules than are shorter wavelengthemitting conjugates. Furthermore, it is easier to work in the red end ofthe spectrum rather than in the ultraviolet region because plasticmaterials do not absorb and emit in the yellow to red spectral region.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1

As an example of a fluorescent conjugate of the invention, aphycoerythrin-immunoglobulin conjugate was prepared. Thiolatedphycoerythrin (PE-SH) was prepared by the addition of 2-iminothiolane tophycoerythrin. Activated immunoglobulin (IgG-S-S-Pyr) containing2-pyridyl disulfide groups was prepared by the addition ofN-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The fluorescentconjugate (PE-S-S-IgG) was then formed by mixing PE-SH with IgG-S-S-Pyr.The product was analyzed by high pressure liquid chromatography (HPLC)on a Varian G3000SW column. This gel filtration column separatesmolecules primarily according to their hydrodynamic radii. PE-SH elutes12 minutes after injection and IgG-S-S-Pyr elutes at about 13 minutes.See FIG. 1 showing the HPLC data. The reaction product PE-S-S-IgGemerges from the column at 8.5 minutes, much sooner than either reactantbecause the conjugate is larger than either component. The fluorescenceemission of a 0.5 ml sample of this conjugate could readily be detectedat a phycoerythrin conjugate concentration of less than 10⁻¹⁰ M.

EXAMPLE 2

A second example of the joining of a phycobiliprotein to anotherbiomolecule is provided by the synthesis of a phycoerythrin-avidinconjugate. Avidin was activated by the addition of m-maleimidobenzoylN-hydroxysuccinimide ester (MBS). The ester group of MBS reacted withnucleophiles on avidin. Sulfhydryl groups on thiolated phycoerythrinthen reacted with maleimide groups on activated avidin molecules.Uncombined avidin was removed from the reaction mixture bychromatography on carboxymethyl-Sephadex.

EXAMPLE 3

A third example of the joining of a phycobiliprotein to anotherbiomolecule is provided by an alternative route for the synthesis of aphycoerythrinavidin conjugate. Biotinylated phycoerythrin was preparedby reacting phycoerythrin with the N-hydroxysuccinimide ester of biotin.Avidin was added to biotinylated phycoerythrin to form aphycoerythrin-biotin-avidin conjugate (PE-B-A). Excess avidin wasremoved by gel filtration. PE-B-A, which binds very tightly tobiotinylated molecules, was then used as a fluorescent stain in afluorescence-activated cell sorting experiment. Biotinylated monoclonalantibody having specific affinity for immunoglobulin D (IgD) was addedto a mixture of spleen cells. This monoclonal antibody combines with IgDmolecules, which are present on the surface of about 40% of spleencells. Excess antibody was removed by washing. PE-B-A was then added tothis mixture of cells. The avidin unit of this highly fluorescentconjugate combined with biotin groups on cell surfaces bearing anti-IgGimmunoglobulin. The fluorescence-activated cell sorter analysis of thiscell population is shown in FIG. 2. The fluorescence intensity of cellslabeled by the phycoerythrin conjugate is comparable to that obtainedwith a fluorescein conjugate in a parallel experiment. This findingdemonstrates that phycobiliprotein conjugates are effective longwavelength fluorescent labels for fluorescence analyses of cells.

EXAMPLE 4

The phycoerythrin-biotin-avidin conjugate described above was also usedto fluorescent-stain beads containing an antigen. Biotinylatedmonoclonal antibody having specific affinity for a target immunoglobulinwas added to agarose beads (insoluble matrices) containing covalentlyattached target antigen. These beads were washed and PE-B-A was thenadded. Beads labeled with this fluorescent phycobiliprotein conjugatewere examined by fluorescence microscopy. The labeled beads appearedyellow when viewed with a standard filter combination designed forfluorescein emission. With longer wavelength filters, the labeled beadsappeared orange-red. A mixture of fluorescein-avidin labeled beads andPE-B-A labeled beads were also examined by fluorescence microscopy. ThePE-B-A labeled beads could readily be distinguished from the fluoresceinlabeled beads because they were yellow rather than green (FIG. 3a) usingfluorescein optics. With a longer wavelength set of filters, only thePE-B-A beads were brightly stained, in this case orange-red (FIG. 3b).These experiments show that phycobiliprotein-biomolecule conjugates areeffective fluorescent stains for fluorescence microscopy.

EXAMPLE 5 Preparation of Phycobiliproteins

R-phycoerythrin was purified from red algae, Gastroclonium coulteri(Rhodymeniales), which were collected from Stillwater Cove, MontereyPeninsula, Calif. The fresh algal tissue was washed with distilledwater, suspended in 50 mM sodium-phosphate buffer at pH 7.0, and blendedfor 3min at the highest speed setting of an Osterizer blender. Thehomogenate was filtered through several layers of cheese cloth andresidual particulate matter removed by low speed centrifugation. Thesupernatant was brought to 60% of saturation with solid (NH₄)₂ SO₄. Allof the above steps were carried out at 4° C. The precipitate wascollected by centrifugation, resuspended in 60% of saturation (NH₄)₂ SO₄in 50 mM sodium phosphate, pH 7.0, and slurried with DEAE-cellulose(microgranular; Whatman, Inc., Chemical Separation Div., Clifton, N.J.).The slurry was packed into a column. The column was developed stepwisewith decreasing concentrations of (NJ₄)₂ SO₄ in 50 mM sodium phosphate,pH7.0, down to 10% of saturation. At that point elution was completedwith 200 mM sodium phosphate, pH7.0. The phycoerythrin eluted between10% saturation (NH₄)₂ SO₄ -50 mM sodium phosphate, pH7.0 and 200 mMsodium phosphate, pH7.0. The eluate was concentrated by (NH₄)₂ SO₄precipitation, re-dissolved in 50 mM sodium phosphate, pH7.0 and (NH₄)₂SO₄ added to 10% of saturation at 4° C. The protein crystallized underthese conditions upon standing at 4° C.

Synechococcus 6301 C-phycocyanin (Glazer and Fang, Biol. Chem. (1973)248:65-662) Anabaena variabilis allophycocyanin (Bryant et al, Arch.Microbiol. (1976) 110:61-75) and B-phycoerythrin (Glazer and Hixson, J.Biol. Chem. (1977) 252:32-42) were prepared as described in thereferences cited.

EXAMPLE 6 Preparation of Phycoerythrin-Avidin

A 50μl aliquot of 1 mg/ml N-hydroxysuccinimidobiotin (Sigma ChemicalCo., St. Louis, Mo. or Biosearch, San Rafael, Calif.) indimethylsulfoxide was added to lml of 2.7 mg/ml R-phycoerythrin (orB-phycoerythrin) in 50 mM sodium phosphate, pH 7.5 to give areagent/phycoerythrin molar ratio of 13. The use of avidin and biotin inlabeling studies has been described previously (Green, Adv. ProteinChem. (1975) 29:85-133; Heitzmann and Richards, PNAS USA (1974)71:3537-3541). After 90min at room temperature, the reaction wasquenched by the addition of 10μl of 100 mM glycyl-glycine, pH 7.5 andthen dialyzed for 3 d at 4° C. against 50 mM sodium phosphate, pH 6.8lml of this mixture of biotinylated phycoerythrin (Biot-PE) andunmodified phycoerythrin was added slowly with stirring to lml of 5mg/ml avidin in the same buffer. The molar ratio of tetrameric avidin tophycoerythrin was 20. This mixture of phycoerythrinavidin conjugates(PE-avidin), phycoerythrin, and avidin was fractionated by high-pressureliquid chromatography.

EXAMPLE 7 Preparation of Phycoerythrin-Immunoglobulin G (PE-IgG)

Thiolated phycoerythrin was prepared by the addition of 600μl of 15.5mg/ml iminothiolane hydrochloride (Sigma Chemical Co.) (Jue et al.,Biochemistry (1978) 17:5399-5406) to 1.2 ml of 3.6 mg/ml R-phycoerythrinin 125 mM sodium phosphate, pH 6.8. After 90min at room temperature, thereaction mixture was dialyzed overnight at 4° C. against 50 mM sodiumphosphate, pH 6.8 and then for 2d against pH 7.5 buffer. Titration of analiquot with 5,5'-dithiobis-(2-nitrobenzoic acid) showed that theaverage content of sulfhydryl groups per phycoerythrin molecule was 8.

A 30μl pf 1.1 mg/ml N-succinimidyl 3-(2-pyridylthio)-propionate (SPDP)(Pharmacia Fine Chemicals, Piscataway, NJ) (Carlsson et al., Biochem. J.(Tokyo) (1978), 173:723-737) in ethanol was added to 700μl of 4.2 mg/mlimmunoglobulin G in 50 mM sodium phosphate pH 7.5. The immunoglobulinwas a monoclonal .sub.γ 1 mouse anti-allotype antibody havingspecificity for the a allotype of the mouse .sub.γ 2a subclass. Themolar ratio of SPDP to IgG was 5.3. The reaction was allowed to proceedfor 2.5h at room temperature. Thiolated phycoerythrin (400μl of 1.7mg/ml in the same buffer) was added to 500μl of this reaction mixture.The molar ratio of activated IgG to thiolated phycoerythrin was 4.7.After 12h at room temperature, 100μl of 80 mM sodium iodoacetate wasadded to block any remaining sulhydryl groups.

EXAMPLE 8 Preparation of Phycoerythrin-Protein A

To 0.5 ml B-phycoerythrin (4.08 mg/ml) in 0.1 M sodium phosphate-0.1 MNaCl, pH7.4, 10μl of SPDP in anhydrous methanol (2.65 mg SPDP/ml) wasadded to give an SPDP/protein molar ratio of 10. The reaction wasallowed to proceed at 22° C. for 50min and was terminated by applyingthe reaction mixture to a column of Sephadex G-25 (1.0×17cm)equilibrated with 100 mM sodium phosphate-0.1 M NaCl, pH 7.4. Thephycoerythrin peak, eluted with the same buffer, was collected andstored at 4° C.

To 0.5 ml protein A (Sigma Chemical Co.) from Staphylococcus aureus, 2mg/ml in 100 mM sodium phosphate-100 mM NaCl, pH 7.4, 2.6μl of the abovemethanolic SPDP solution was added to give an SPDP/protein molar ratioof 9.5. After 40min at 22° C, the reaction was terminated by theaddition of 25μl of 1 mM dithiothreitol in the pH 7.4 buffer. After25min at 22° C, the reaction mixture was subjected to gel filtration asdescribed above and the protein A peak collected.

Appropriate volumes of the phycoerythrin-S-S-pyridyl derivative and ofthe thiolated protein A were mixed to give a molar ratio ofphycoerythrin to protein A of 1:2. The reaction mixture contained 0.77mg phycoerythrin/ml and 0.27 mg of protein A/ml. After 6h at 22° C., thereaction mixture was stored at 4° C. and the phycoerythrin-protein Aconjugate generated in this manner was used without furtherpurification.

EXAMPLE 9 Preparation of Target Beads

IgG-Sepharose beads were prepared by adding 1.2 ml of 5.2 mg/ml IgG to 2ml of a slurry of CNBr-activated Sepharose 4B (Pharmacia FineChemicals). The immunoglobulin was a mouse .sub.γ 2a myeloma antibody ofthe a allotype. After 2h of end-over-end mixing at room temperature, thereaction was quenched by the addition of 2 ml of 1M glycine, pH 7.5. Thebeads were then washed exhaustively. Ovalbumin-Sepharose beads andavidin-Sepharose beads were prepared similarly. Biotin-Sepharose beadswere prepared by reacting ovalbumin-Sepharose beads withN-hydroxysuccinimidobiotin.

Spectroscopic Measurement

Absorption spectra were obtained on a Beckman model 25 spectrophotometer(Beckman Instruments, Inc., Fullerton, Calif.). Fluorescence spectrawere obtained on a Perkin-Elmer model 44B fluorometer equipped withDCSCU-2 corrected emission spectra unit, or on a Spex Fluorologinstrument. Fluorescence microscopy was carried out using a ZeissUniversal microscope with epiillumination optics.

High-pressure Liquid Chromatography

Coupling reactions were followed by high-pressure liquid chromatographyon a Waters instrument with a Varian G3000SW gel filtration column whichseparates molecules primarily according to their hydrodynamic radii. Foranalyses, 20μg protein was applied in a volume of 10-20μl. Inpreparative experiments, 750μl of sample was applied and 400μl fractionswere collected. The eluting-buffer was 200 mM sodium phosphate, pH 6.8and the flow rate was 1 ml/min.

Fluorescence Staining of Lymphocytes

Human peripheral blood leukocytes were prepared using Ficoll-Hypaquegradients. Viability counts of the cells recovered were done by stainingthe cells with acridine orange and ethidium bromide and countingfluorescent cells with a fluorescence microscope using standardfluorescein optics. This dye combination stains live cells green anddead cells orange-red. Cell preparation were always >95% viable.

The anti-Leu antibodies used in this study were all monoclonally derivedhybridoma antibodies (Becton, Dickinson & Co. Monoclonal Center,Mountain View, Calif.). The green fluorescence signal came from directlyfluoresceinated antibodies. The red fluorescence signal came frombiotinylated antibodies counter-stained with phycoerythrin-avidin.Fluorescent antibody staining was done in one or two steps. Directlyfluoresceinated antibodies were incubated with 10⁶ cells in 50μl ofHEPES-buffered (10 mM) RPMI-1640 medium (deficient in phenol red andbiotin) containing 5% (vol/vol) horse serum and 0.2% (wt/vol) azide for20min on ice. The amount of antibody added had been previouslydetermined to be optimal for staining this number of cells. Fortwo-color staining, both the directly fluoresceinated antibody and thebiotinylated antibody were incubated with 10⁶ cells in 50μl of mediumfor 20min on ice. After washing the cells twice with medium, thephycoerythrin-avidin conjugate was added to the cells in 50μl of medium.This mixture was incubated for an additional 20min on ice before finallywashing the cells three times in medium. Cells were resuspended in 0.5ml of medium for fluorescence analyses using the fluorescence-activatedcell sorter.

Fluorescence-activated Cell Analyses

A modified Becton, Dickinson & Co. Fluorescence-activated Cell Sorter(FACS II) was used for fluorescence analyses of single cells. A 560 nmdichroic mirror divided the emission into a shorter wavelength component(the "green" channel) and a longer wavelength component (the "red"channel). Logarithmic amplifiers with a 3.5 decade logarithmic rangewere used for both channels Electronic compensation (Loken et al., J.Histochem. Cytochem. (1977) 25:899-907) corrected for fluoresceinspillover into the red channel and for phycoerythrin spillover into thegreen channel. These corrected signals will be referred to as green andred fluorescence. The fluorescence data was displayed as contour mapswhich resemble topographic maps. The contour lines depict cell densityon a linear scale. The number of cells in a region of the map isproportional to the volume represented by the contours in that region.The computer program that acquires and displays FACS data in this mannerwas written by Wayne Moore (Stanford University).

PE-avidin conjugate was prepared by biotinylating phycoerythrin and thenadding an excess of avidin. An average of one biotin per phycoerythrinwas incorporated when a 13-fold molar excess ofN-hydroxysuccinimidobiotin was reacted with phycoerythrin for 90min. Thereaction mixture obtained upon subsequent addition of a large excess ofavidin was analyzed by high-pressure liquid chromatography.Phycoerythrin-avidin conjugates eluted first, followed by phycoerythrinand biotinylated phycoerythrin and then by avidin, as expected on thebasis of their hydrodynamic radii. The reaction mixture contained asubstantial amount of PE-avidin conjugates (peaks 1 and 2), in additionto phycoerythrin (peak 3) and avidin (peak 4). It seems likely that peak2 corresponds to a conjugate containing one avidin molecule joined toone molecule of biotinylated phycoerythrin, whereas peak 1 and theregion between peaks 1 and 2 consist of conjugates made up of three ormore protein molecules. Fractions corresponding to peak 2 were collectedand pooled. The major species is PE-avidin. Some unreacted phycoerythrinand a small amount of untreated avidin remained after this fractionationstep. The capacity of this PE-avidin preparation for biotin was testedby adding it to biotin-Sepharose beads. These stained beads displayedintense orange-red fluorescence characteristic of phycoerythin. Theprior addition of avidin to biotin-Sepharose beads, or of biotin to thePE-avidin conjugate, blocked the binding of PE-avidin to these beads, asevidenced by the fact that they appeared dark under the fluorescencemicroscope. Likewise, biotin-Sepharose beads stained with eitherphycoerythrin or biotinylated phycoerythrin did not fluoresce in theorange-red spectral region. These experiments, as well as thefluorescence-activated cell analyses to be discussed shortly,demonstrate that the PE-avidin conjugate binds specifically to biotinand biotinylated molecules. Furthermore, the quantum yield and emissionspectrum of the PE-avidin conjugates are virtually the same as those ofnative phycoerythrin. A 1 ml sample of 10-12 M phycoerythrin (orphycoerythrin conjugate) excited at 520 nm gives a fluorescence signalat 576 nm that is twice as large as the Raman scattering at 631 nm fromwater. Thus, 10-15 mole of phycoerythrin in a standard fluorescencecuvet can readily be detected.

It is of interest to compare the fluorescence intensity of phycoerythrinwith that of fluorescein. The fluorescence intensity of a dilutesolution of a chromophore is proportional to cεQ, where c is the molarabsorbance coefficient at the excitation wavelength, and Q is thefluorescence quantum yield. For excitation ar 488 nm (an argon-ion laserline): ε=2.18× 10⁶ cm⁻¹ M⁻¹ and Q=0.82 for phycoerythrin; and ε=8× 10⁴cm⁻¹ M⁻¹ and Q =0.9 for fluorescein. Hence, a solution of phycoerythrinexcited at 488 nm has a fluorescence intensity 14.5 as high as that ofan equimolar solution of fluorescein. The observed intensity ratiodepends also on the efficiency of the detection system with respect toemission wavelength. A phycoerythrin/fluorescein intensity ratio of 10was measured when equimolar solutions of these substances were flowedthrough the cell sorter. It is estimated that 10³ molecules ofphycoerythrin bound to a cell could be detected in flow cytometryexperiments.

IgG-Sepharose beads displayed bright orange-red fluorescence afterstaining with the phycoerythrin-protein A conjugate, as anticipated,since protein A is known to bind to the F_(c) portion of mouse 2aimmunoglobulins. This staining of the beads was inhibited by theaddition of the soluble IgG2a. Likewise, the conjugate of phycoerythrinwith monoclonal anti-allotype antibody stained Sepharose beadscontaining covalently attached target immunoglobulin. These beads didnot become fluorescent if soluble target immunoglobulin was added firstto the phycoerythrin-antibody conjugate, or if soluble anti-allotypeantibody was added first to the beads. Thus, the specific bindingcharacteristics of the protein and of the anti-allotype antibody wereretained in their conjugates with phycoerythrin.

The PE-avidin conjugate was used as the red-fluorescent stain intwo-color fluorescence-activated cell analyses. The distribution of Leuantigens (OKT antigens) on the surface of human T-lymphocytes wasinvestigated. (The identity of Leu and OKT antigens has recently beendetermined. Leu-1=OKTl; Leu-2=OKT8; Leu-3=OKT4; and Leu-4=OKT 3.) Leu-1and Leu-4 are known to be present on all T-cells, whereas Leu-2a andLeu-2b are associated with suppressor and cytotoxic T cells, and Leu-3aand Leu-3b with helper and inducer T cells. The densities of Leu-1,Leu-2a, Leu-3b, and Leu-4 were determined by staining cells with afluoresceinated antibody specific for one of these antigens andmeasuring green fluorescence of these cells. The density of Leu-3a wasascertained by staining T-lymphocytes with biotinylated antibodyspecific for Leu-3a, followed by PE-avidin, and measuring the redfluorescence. In a control experiment, the cells were stained withPE-avidin, but not antibody. The peak near the origin of this plotarises from the autofluorescence of these cells, inasmuch as unstainedcells give the same pattern. Thus, PE-avidin has virtually no affinityfor these cells. The result of staining these cells with fluoresceinatedanti-leu-3b was two peaks, one near the origin arising from cells (˜50%)devoid of Leu-3b on their surface, and the other coming from cells(˜50%) expressing Leu-3b. The green signal is 50 times as bright as theautofluorescence of the Leu-3b negative cells, whereas their red signalsare the same. The converse result, was obtained when T-lymphocytes werestained with biotinylated anti-Leu-3a, followed by PE-avidin. The redfluorescence of cells containing Leu-3a on their surface is 30 times ashigh as that of negative cells, whereas the green signals of these twopopulations are the same. The result of a simultaneous analysis forLeu-3a and Leu-3b is a peak near the origin arising from cells thatexpress neither antigen, and another peak which comes from cells thatcontain both Leu-3a and Leu-3b. In other words, every cell thatexpresses Leu-3a also expressed Leu-3b, and vice versa. This binding isexpected, because 3a and 3b are known to be nonoverlapping determinantson the same protein molecule.

Two-color fluorescence-activated cell analyses of the distribution ofLeu-3a relative to Leu-1, Leu-2a, and Leu-4 on human peripheral bloodlymphocytes were also carried out. The observed Leu 1 staining patternindicates that ˜60% of peripheral blood lymphocytes from this donor areT-cells because the Leu-1 antigen is known to be present on all humanperipheral T-cells. The profile obtained by also staining for Leu-3ashows that not all T-cells bear the Leu-3a antigen. About 80% of theT-cells carry Leu-3a and Leu-1. These doubly-positive cells include thehelper and inducer T-cell subpopulations. A different result wasobtained for the distribution of Leu-2a and Leu-3a. Only 10% of alllymphocytes analyzed express Leu-2a an antigen known to be associatedwith the suppressor and cytotoxic T-cell subpopulations. The profile forLeu-2a and Leu-3a demonstrates that a T-cell expresses Leu-2a or Leu-3abut not both antigens. This mutual exclusion is in harmony with the factthat a particular T-cell may be either a suppressor-cytotoxic or ahelper-inducer cell, but not both. The distribution of Leu-4 is similarto that of Leu-1, as expected because Leu-4 is known to be present onall human peripheral T-cells. In staining for Leu-3a and Leu-4 all cellsthat were found positive for Leu-3a are also positive for Leu-4.

The above data demonstrate that biliprotein conjugates are a valuableclass of fluorescent reagents for analyses of molecules. Thefluorescence-actived cell sorter examples are specific evidence of theutility and variety of analyses which one can carry out employing thebiliproteins. The high extinction coefficient of phycoerythrin at 488nm, the position of an argon-ion laser line, makes it possible tosimultaneously excite fluorescein and phycoerythrin with highefficiency. Their fluorescence emission maxima lie at 515 and 576 nm,respectively, and so their emission contributions can readily beseparated by appropriate filters. An advantage of using phycoerythrin isthat a single laser line suffices for two-color analyses.

The phycobiliprotein conjugates open up a possibility of carrying outthree-parameter analyses with two laser sources. For example,allophycocyanin could serve as the third fluorescent chromophore. Insuch a three-color experiment, fluorescein and phycoerythrin could beexcited by the 488 nm argon-ion line and allophycocyanin by the 625 nmoutput of a dye laser (or by a krypton or helium-neon laser). Theabsorption and emission spectra of phycobiliproteins point to thepossibility of four-color analyses if C-phycocyanin conjugates were alsoemployed. The phycobiliproteins are well suited for fluorescenceimmunoassays. The fluorescence of femtomole quantities ofphycobiliproteins such as phycoerythrin can readily be detected.Furthermore, background fluorescence from body fluids and supportingmedia, diminishes markedly in going to the red end of the spectrum. Theorange-red emission of phycobiliproteins is particularly advantageous inthis regard. Furthermore, the phycobiliproteins can be conjugated to awide variety of ligands and receptors without interfering with thefunctioning of the ligand or receptor in specific binding pairs, norlosing the desired spectral properties of the phycobiliproteins.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A receptor covalently bound to aphycobiliprotein.
 2. A receptor according to claim 1, wherein saidreceptor is a cell surface protein receptor.
 3. A receptor according toclaim 1, wherein said receptor is avidin.
 4. A receptor according toclaim 1, wherein said receptor is an immunoglobulin.